In this study, a sandwich immunoassay method utilizing enzymatic activity of alkaline phosphatase (ALP) on 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for Escherichia coli (E. coli) detection was developed using surface enhanced Raman spectroscopy (SERS). For this purpose; spherical magnetic gold coated core-shell nanoparticles (MNPs-Au) and rod shape gold nanoparticles (Au-NRs) were synthesized and modified for immunomagnetic separation (IMS) of E. coli from the solution. In order to specify the developed method to ALP activity, Au-NRs were labeled with this enzyme. After successful construction of the immunoassay, BCIP substrate was added to produce the SERS-active product; 5-bromo-4-chloro-3-indole (BCI). A good linearity (R-2 = 0.992) was established between the specific SERS intensity of BCI at 600 cm(-1) and logarithmic E. coli concentration in the range of 1.7 x 10(1)-1.7 x 10(6) cfu mL(-1). LOD and LOQ values were also calculated and found to be 10 cfu mL(-1) and 30 cfu mL-1, respectively. (C) 2017 Elsevier B.V. All rights reserved.